SUMMARY Cytochrome c (Cytc) plays a central role in mitochondrial respiration and type 2 apoptosis. We have developed new protocols to purify mitochondrial proteins while maintaining their physiological regulatory properties and posttranslational modifications, and we discovered that Cytc is phosphorylated on distinct tyrosine residues in heart and liver tissue. Recently, we have mapped two more phosphorylation sites on Cytc purified from cow and rat kidneys, Thr28 and Ser47, which are the primary focus of this grant application. Supported by strong preliminary data it is our overall hypothesis that Cytc phosphorylation regulates the two main functions of Cytc, mitochondrial respiration and apoptosis. Our long term goal is to understand the regulation of Cytc by cell signaling pathways under normal and pathological conditions. As a first step towards our goal we will test four specific hypotheses: 1) to test the hypothesis that kidney Cytc can be phosphorylated on Thr28 and Ser47, and that these modifications affect the basic properties of the molecule; 2) to test the hypothesis that Cytc Thr28 and Ser47 phosphorylation affects mitochondrial respiration and apoptosis; 3) to identify kinases and phosphatases that control Cytc phosphorylation; and 4) to demonstrate the physiological effect in mice. Phosphorylated Cytc will be isolated from cow and rat kidneys and phosphomimetic mutant Cytc will be overexpressed and purified from bacteria, followed by structural characterization using mass spectrometry, spectroscopic methods, and protein crystallography (Aim 1). Phosphorylated and phosphomimetic mutant Cytc will be subjected to a comprehensive set of functional analyses including in vitro respiration and apoptosis measurements, and by in vivo studies with phosphomimetic Cytc stably expressed in Cytc knockout cells (Aim 2). We will next lay the ground work to explore the signaling pathways and identify kinases and phosphatases that act on Cytc using affinity purification/mass spectrometry techniques (Aim 3). Finally, we will reintroduce phosphomimetic and non-phosphorylatable mutant Cytc into Cytc knockout mice to study the effect of Cytc modification and regulation at the animal level (Aim 4). We expect that this research will reveal that Cytc, a protein that makes life and death decisions, is subject to regulation by cell signaling, opening new opportunities for the understanding of mitochondrial respiration and apoptosis, and to control it in pathological conditions in which respiration and apoptosis are dysregulated.